This is a 371 of PCT/FR98/01213 filed on Jun. 11, 1999.
The field of the invention is that of the detection of products, preferably xe2x80x9caffinityxe2x80x9d biological products, such as nucleic acids, or else biopolymers of a protein nature.
More specially, present invention relates, on the one hand, to a method for the qualitative and/or quantitative analysis of biological substances CBS, preferably polynucleotides PN present in a conductive liquid (solution or gel) medium CL, by means of optoelectrochemical measurements and, on the other hand, to the devices and the affinity sensors intended for implementing this method.
The biological substances more particularly, but not exclusively, for which the invention is targeted are polynucleotides PN. This general term encompasses, according to the invention, molecules composed of at least two nucleotides (oligonucleotides and polynucleotides stricto sensu), including especially the nucleic acids RNA or DNA and any genetic structure comprising them. The invention also relates to the compounds that can be involved in immunological coupling [antigen Ag/antibody Ab] reactions or even Enzyme/Substrate E/S recognition reactions.
In order to detect, identify or quantitatively determine these molecules, their bioaffinity properties. have been exploited, namely their particular ability to specifically pair with their complementary species, according to Ag/Ab immunological coupling or E/S recognition PN/cPN genetic hybridization mechanisms. Thus, in the immunological field, methods are known which are based on antigen/antibody coupling and which involve a step of revealing the pairs formed using radioactive, enzyme, fluorescent, colored or similar labels. Such methods are lengthy and complex to implement. In addition, the reactants used are not commonly available and are expensive. Finally, these methods do not allow continuous measurements and even less in vivo measurements. These techniques have been transposed with their shortcomings to the field of the detection of nucleic sequences, labeled in order to be able to be identified and/or quantitatively determined.
In another context, it has been proposed to detect physical signals of any kind that are capable of being induced by nucleotide-hybridization or immunological coupling biochemical reactions. To do this, it is appropriate, first of all, to isolate one type of particular and characteristic signal and then to use a transducer capable of converting said signals into a measurable physical quantity. These signals, may, for example, be produced by a chemical species, a variation in thickness, in optical index or in mass, or even a variation in electrical properties. The transducers may therefore be electrochemical, piezoelectric, optical or electrical sensors. The entire difficulty resides in how to bring out the specific signals from the pairing and how to develop a corresponding transducer which is reliable, sensitive and reproducible.
The invention described in French Patent Application No. 94/08688 falls perfectly within this prior art based on the detection of electrical signals induced by PN/cPN hybridizations or by antigen-antibody reactions, in an electrically conductive liquid medium.
That patent application describes a method for qualitatively and/or quantitatively analyzing biological substances, particularly polynucleotides, antigens, antibodies, enzymes and substrates, in which a multilayer structure comprising a semiconductor wafer is used, said wafer being covered with an insulator whose surface is functionalized by one of the species of the pairs of specifically pairable biological substances mentioned above. According to that method, the semiconductor containing polynucleotides PN to be quantitatively determined or to be identified are biased and the variations in the electrical signals induced by a charge effect phenomenon, directly and essentially related to the pairings of the intended biological substances with their complementary ligands immobilized on the insulator, possibly by means of a sensitive membrane, are collected.
This electrical transduction measurement technique does not require a reaction intermediate, a specific label, or an enzyme reaction. It gives satisfactory results but it nevertheless remains to be improved with regard to simplicity of implementation and capability of taking a series of rapid measurements on various substrates, without using as many sensors as there are different kinds of substrates to be analyzed. According to that technique, the charge-effect phenomenon may be apprehended by measuring the electrochemical impedance of a semiconductor/-insulator/sensitive membrane/conductive liquid structure. According to one embodiment, the field effect due to the variation in the surface charge induced by the pairing may be characterized by means of a biased field-effect transistor on which the variations in the gate/source potential induced by the field effect are measured.
Moreover, technical proposals have been made for the identification and/or quantitative determination of biological molecules, which combine
on the one hand, means for revealing the substances to be quantitatively determined, using radioactive, enzyme, fluorescent or colored labels, redox-potential or pH modifiers, or similar means, and
on the other hand, electrochemical or optoelectrochemical means of transduction.
Unfortunately, such a combination is not of the type to eliminate the drawbacks associated with the revelation-based techniques, nor to improve the electrochemical analysis techniques.
Thus, U.S. Pat. Nos. 4,591,550 and 5,500,188 disclose a method and a device for the determination and quantitative analysis of one or more substances which are contained in a gaseous or solid liquid medium and are capable of modifying the photoresponse characteristics of a photosensitive element, comprising means for recognizing said substances. These means involve a mechanism of revelation by one or more tracer substances capable of modifying the physicochemical characteristics of the analysis medium (pH or redox potential) and/or capable of being distinguished by a radioactive, colored or fluorescent label. It is therefore a hybrid technique which combines optoelectrochemical means of transduction with physicochemical means of revelation.
The device employed in that known method comprises one or more sensors each consisting of a semiconductor wafer (silicon covered with an SiO2 or silicon nitride insulator layer), the surface of the latter possibly being functionalized by means for recognizing the substances to be identified and/or quantitatively determined. That device is also provided with means for biasing the semiconductor/insulator structure (e.g. a circuit for applying a biasing voltage, said circuit comprising, on the one hand, a counterelectrode and a reference electrode and being connected, on the other hand, to the semiconductor/insulator structure) The device furthermore includes means for irradiating the photosensitive element or elements as well as means for measuring the resulting electrical signals, for detecting and/or for identifying the substances in question.
It should be noted that each photosensitive element comprising the semiconductor/insulator structure is necessarily associated with illumination means, biasing means and measurement means. It follows that the device is extremely complex in its alternative forms aimed at the multiple detection of different substances, both with regard to the structure as it is and to the taking and processing of the measurements and of the resulting signals.
According to this prior art, the insulator layer of the photosensitive elements is functionalized if the substances to be analyzed are PN/cPN, E/S or Ag/Ab affinity systems. In this case, the recognition ligands functionalizing the insulator layer are systematically labeled. This is illustrated especially in the case of DNA or RNA analysis, in which the complementary recognition ligands are labeled with biotin (cf. column 14, lines 49 to 63 of U.S. Pat. No. 5,500,188). In such configurations, all the drawbacks associated with these revelation techniques using a label must therefore be borne. Moreover, it should be pointed out that those techniques involving the absorption or emission of light radiation run the risk of further disturbing the intended activation irradiation according to this method.
If the systems are not affinity systems involving specific functionalization of the insulator of the sensors, the tracing means are, for example, variations in pH or in redox potential. These means should be considered as being not the most reliable since there are a good number of factors in the analysis medium, which is for example a conductive liquid, which can interfere with these parameters without this being connected with the identification and quantitative determination of the intended substances.
In addition, although the fact that the measured signal output by their sensors may be the photopotential, the photoconductance, the photocapacitance or the photoinductance, or combinations of these (column 3, lines 36 and 37 of U.S. Pat. No. 5,500,188) is not excluded, it is mentioned, in lines 41 to 43, column 3 of this same patent, that the measured signal is the resultant of a change in a DC current, of an AC current or of the effect of a DC current on an AC current. This preference for taking into account the photocurrent as the resulting signal is also clearly, and exclusively apparent from the examples of these U.S. patents which take into account either the current needed to maintain a constant potential between the sensitive element and the reference (column 19, lines 4 to 7), or the variations in this current, which correspond to the changes in the chemical environment in the region of the insulator of the sensitive element (column 19, lines 43 to 45), or the potential required for maintaining a constant current (column 19, lines 62 to 65) or the variation in the current supplied to the irradiation means necessary for maintaining a constant potential between the measurement structure and the reference (column 20, lines 42 to 49). This is also apparent from the passages in column 26, lines 7 to 9 and lines 30 to 34.
It is therefore apparent that the main signal taken into consideration in the method and the device according to patents U.S. Pat. No. 4,591,550 and 5,500,188 is the photocurrent measured either directly or indirectly. However, the relationship between the illumination and this photocurrent is poorly defined and involves several physical phenomena. This therefore results in uncertainty in the interpretation of the measurements.
It therefore has to be stated that this hybrid technical proposal mentioned above is in no way satisfactory in terms of the qualitative and/or quantitative analysis of substances, particularly biological substances, and even more particularly polynucleotide substances. Moreover, the Applicants wish to point out as proof of this that, there is at the present time on the market no industrial and commercial application of the invention described in these patents U.S. Pat. Nos. 4,591,550 and 5,500,188.
Thus, in order to present the problematic at the heart of the present invention, the inventors are especially tasked, as essential objective, to provide a method for the identification and quantitative determination of biological substances, preferably polynucleotides, this method having to incorporate especially the following specifications:
specificity;
high sensitivity;
ease of implementation;
xe2x80x9capplicabilityxe2x80x9d to a wide variety of biological substances, and especially polynucleotide substances;
low manufacturing costs;
possible miniaturization so as to allow in situ and/or in vivo analytical measurements, continuously or intermittently;
high reliability,
good reproducibility;
possibility of multiple detection, that is to say the identification and/or quantitative determination of different substances (heteroclite polynucleotides) contained in one and the same analysis medium;
application to the diagnosis of viral or genetic disorders, since it involves multiple detection which is simple to implement and interpret with respect to polynucleotides.
To meet these objectives, among others, the inventors have to their credit demonstrated, very surprisingly and unexpectedly, that the specific pairings between molecules, preferably biological molecules, and even more preferably between complementary polynucleotide strands, modify the surface electric charge in a multilayer-semiconductor Sc/surface-functionalized insulator Is-structure. This modification occurs more specifically at the interface with a conductive liquid medium CL, said charge effect constituting the base signal for identification and quantitative determination perceived, directly or indirectly, by optoelectrochemical transduction means.
The way in which this novel and advantageous concept is put into practice is expressed through the present invention, the subject of which is therefore a method for the identification and/or quantitative determination of Biologicalxe2x80x94preferably electrically Charged Biologicalxe2x80x94Substances (CBS) present in a Conductive Liquid medium CL, using at least one affinity sensor comprising at least one structure having at least one semiconductor material Sc coated on at least one of its faces with at least one layer of insulator Is, this layer having on its surface at least one probe Pr, in contact with the conductive medium CL and comprising one or more Recognition Biological Substances (RBS) for the specific recognition, by affinity interaction, of the CBSs in the abovementioned medium CL, which essentially consists in:
-a- selecting, as probe(s) Pr, unlabeled RBSs;
-b- taking measures to ensure that the Fermi level of the Sc corresponds approximately to, or passes by, the intrinsic level at the surface of the Sc;
-c- exposing the Sc to a periodic illumination comprising photons whose energy is greater than or equal to the band gap energy of the Sc;
-d- measuring, directly or indirectly, the variations xcex94Vfb in the flat band potential Vfb of the Sc, which are induced by a charge-effect phenomenon directly and essentially associated with the specific pairings of the CBSs in the conductive medium CL with their complementary ligands RBS of the probe or probes Pr, to the exclusion:
(i) of the variations resulting from possible charge and/or charge-transfer effects caused by enzyme-catalyzed chemical reactions in which some of the substances to be detected are consumed,
(ii) and of the variations in the photoresponse which are associated with the appearance in the medium CL of at least one tracer product capable of being revealed through pH variations or redox potential variations and/or through labels, preferably of the type of those absorbing or emitting radiation (for example, fluorescent, radioactive or colored labels);
-e- and interpreting the received signals in terms of identification and/or quantitative determination of the CBSs in the CL.
Such a mode of measurement, by optoelectrochemical transduction, meets the desired specifications of simplicity, sensitivity, specificity, reliability and reproducibility.
Moreover, the technique according to the invention also has the advantage of being reversible. This is because it is readily possible to unpair the complementary species that have specifically reacted at the sensitive membrane of the semiconductor structure. The sensitive membrane may thus be regenerated after each use and this can be repeated many times.
Such advantageous results were a priori difficult to be able to predict.
In particular, this technique has proved to be remarkably effective in the context of the recognition of polynucleotide sequences by hybridization of the nucleic acid monostrands (ligands), which form these sequences, with complementary polynucleotide monostrands, immobilized on the layer of insulator of the Sc/Is structure. Thus, according to a preferred mode of implementing the method according to the invention, the CBSs are polynucleotides PN and the RBSs are polynucleotides cPN.
The present invention makes it possible to envisage, in particular, the recognition of nucleotide sequences, for example for the purpose of detecting genetic disorders, for detecting and characterizing viruses, bacteria and parasites, or for establishing genetic maps and for studying the expression and/or mutation of genes.
Apart from nucleotide hybridization, it is conceivable to exploit other specific pairings such as, for example, the biochemical mechanisms of immunological coupling or even of enzyme/substrate complexing, provided that the pairing results in a variation in electric charge at the surface of the Is.
The analytical principle governing the method according to the invention is exclusively optoelectrical or optoelectrochemical. This means that there has to be affinity-type biochemical recognition, that is to say recognition which involves neither chemical nor enzymatic reactions and which takes place without the production or consumption of intermediate chemical species. Furthermore, the revelation of this affinity-type biochemical recognition does not occur through indirect detection using physicochemical means of revelation: colored, fluorescent, radioactive, redox-potential or pH tracers. According to the invention, the biochemical recognition takes place essentially, or even exclusively, by an optoelectronic transduction which involves biasing the Sc/Is structure with respect to the CL, as well as periodically illuminating said structure.
In the context of the invention, the expression xe2x80x9celectrically chargedxe2x80x9d means that in the affinity interaction of the cPN with the PN, the surface electric charge is modified.
The first step -a- of the method according to the invention consists in employing exclusively probes Pr, particularly cPNs, which are not labeled, that is to say they do not carry physicochemical means of revelation (fluorescence, colorimetry, radioactivity, redox potential or pH).
In the case of a structure surface-functionalized by probes consisting of polynucleotide monostrands cPN, for example DNA, the hybridization of the latter with the strands to be identified or quantitatively determined in the medium CL involves an increase in the charges on the surface of the structure. This modifies the distribution of the charge carriers in the space-charge region of the semiconductor so as to satisfy the new thermodynamic equilibrium of the structure. This new distribution is manifested by a modification of the band curvature of the semiconductor in its interfacial region in contact with the dielectric. The flat-band potential Vfb is the potential that has to be applied to the semiconductor with respect to the medium CL (the electrolyte) in contact with the dielectric Is in order for the bands of the Sc to have zero curvature. According to the invention, this physical quantity Vfb or its variation xcex94Vfb is measured so as to characterize the equilibrium state of the structure. Thus, a modification of the surface charge corresponds to a variation of Vfb, which is itself a signature of PN/cPN hybridization.
In order for there to be a variation in the curvature of the bands of the Sc and therefore in Vfb, it is necessary, in accordance with step b of the method according to the invention, to create an initial band curvature of the Sc by adjusting the Fermi levels of the Sc and of the medium CL, preferably by imposing a DC bias voltage Vb on the Sc/Is-cPN structure with respect to the medium CL.
In accordance with a first mode of implementing the method according to the invention, the Fermi level of the semiconductor Sc is adjusted, to approximately its intrinsic level at the surface, by imposing a bias voltage Vb on the Sc with respect to the CL by sweeping between a negative limiting voltage and a positive limiting voltage, these being chosen in such a way that the Fermi level of the Sc passes by its intrinsic level at the surface, Vb thus advantageously varying within a voltage range corresponding to the depletion and weak-inversion regime of the Sc.
This DC biasing is associated, on the one hand, with the periodic illumination according to step c and, on the other hand, with a measurement of the Vfb variations in step d. This measurement is carried out as follows:
detection of the photopotential (Vph) at the terminals of the Sc (between Sc and CL) and/or of the photocurrent (Iph);
calculation of the in-phase component Zop and/or the quadrature component Zoq of the optoelectrochemical impedance for each value of Vb;
construction of the Zop and/or Zoq and/or Vph and/or Iph curve/as a function of Vb;
and monitoring of the shift in this (or these) curve(s) parallel to the x-axis (potential Vb axis), said shift corresponding to the desired variations in Vfb.
The optoelectrochemical impedance components Zop and Zoq are used to obtain the energy properties of the Sc/Is-Pr structure and of the interfacial region between said structure and the CL, and especially to obtain the various charge effects that may occur at the interfaces. These optoelectrochemical impedance components Zop and Zoq are linked to the photopotential Vph and the photocurrent Ihxcexd which are generated following the periodic luminous excitation of the Sc, the latter being, moreover, DC biased. This is because the modulated illumination (step c) of the Sc by photons having an energy greater than or equal to the width of its band gap generates electron-hole pairs or charge carriers. The latter are separated due to the effect of the electric field existing in the space-charge region of the semiconductor Sc (in the region in contact with the dielectric Is). This phenomenon causes the appearance of a periodic Ihxcexd in the semiconductor Sc, resulting, across the terminals of the Sc/Is-Pr structure, in a periodic photopotential Vph or a periodic photocurrent Iph.
It is therefore possible to use, indiscriminately, Vph and/or Iph and/or the impedance components Zop and/or Zoq as transduction parameters of the charge-effect energy phenomena directly associated with the pairings of interest.
If the power of the illumination in step c is low, the optoelectrochemical impedance components Zop and Zoq are transfer functions linking Vph to Ihxcexd induced by the modulated illumination. More specifically, the optoelectrochemical impedance components Zop and Zoq near the space-charge region of the Sc are directly proportional to the effective values of the in-phase and quadrature components of the measured Vph. The coefficient of proportionality depends on the intensity or power of the illumination.
The photopotential is therefore the image of the optoelectrochemical impedance of the structure.
In this version, it is preferred for the illumination to be not only weak but also approximately sinusoidal.
If the illumination employed at step c is strong, the Vfb variations are obtained, in the context of step d, by preferably:
detecting Vph and/or Iph 
constructing the Vph=f(Vb) curve and/or the Iph=f(Vb) curve and
monitoring the shift in this (or these) curve(s) parallel to the x-axis (potential Vb axis), said shift corresponding to the desired xcex94Vfb values. This is because, with strong illumination, if the surface charge varies because of the pairing of the PN in the CL with the cPNs in the probes Pr of the structure Sc/Is, it follows that there is a variation in Vfb manifested by the curve of the effective value of the photopotential Vph and/or of the photocurrent Iph being shifted parallel to the axis on which the bias potentials of the structure are plotted.
According to the invention, the notions of weak and strong illumination are defined as follows:
weak illumination is illumination which results in a second-order perturbation of the structure with respect to the thermodynamic equilibrium in the dark. It is preferably less than or equal to 10 xcexcW/cm2 and preferably less than or equal to 1 xcexcW/cm2;
strong illumination is illumination which greatly perturbs the thermodynamic equilibrium of the structure in the dark. It is preferably greater than 10 xcexcW/cm2.
In the weak- and strong-illumination versions mentioned above, in the case of the description of the first mode of implementing the method according to the invention, the calculations of Zop and/or Zoq, the controlling and acquisition of the measurements, and the plotting of the Zop and/or Zoq and/or Vph and/or Iph=f(Vb) curves are carried out by means of a microcomputer, using procedures known per se (especially calculation of the impedances according to Ohm""s law).
In a second mode of implementing the method according to the invention, the following methodology is adopted:
in step b, the Fermi level of the Sc is set approximately to the intrinsic level at the surface of the Sc, by imposing a bias voltage Vbi corresponding approximately to the abscissa of the point of inflection of the Vph=f(Vb) or Zoq=f(Vb) or Iph=f(Vb) curve, Vph, Zoq and Iph being as defined above (page 9, lines 4 to 7), in such a way that the ordinate of this point of inflection corresponds approximately to Vph,xc2xdmax, Zoq,xc2xdmax or Iph,xc2xdmax;
and in step d, the variations xcex94Vfb in Vfb are obtained:
(i) by measuring the change in Vph and/or Iph and/or Zoq, if the variations xcex94Vfb are small and assuming that Vfb varies linearly with Vph, then xcex94Vfb=Kxcex94Vph, where K represents the slope of the Vph=f(Vb) curve at the point of inflection;
(ii) and/or by taking into account the variations in the bias voltage xcex94Vb made necessary in order to regulate, and keep constant, the Vph,xc2xdmax, the Zoq,xc2xdmax or the Iph,xc2xdmax, this xcex94Vb adjustment being the reflection of xcex94Vfb.
It is therefore a question, according to this mode of impementation, of biasing the Sc/Is structure in such a way that the Fermi level is in the vicinity of the intrinsic position of the semiconductor. The imposed potential Vbi is close to the potential corresponding to the point of inflection of the sigmoidal-shaped curve of the photopotential Vph, of the photocurrent Iph or the quadrature optoelectrochemical impedance component Zoq as a function of the bias potential Vb.
In the context of the invention, the expression xe2x80x9cin the vicinityxe2x80x9d of the Fermi level with respect to the intrinsic level at the surface of the Sc should be understood to mean the position close to the middle of the band gap of the semiconductor at its surface in contact with the insulator Is, within a range such that the semiconductor at the surface is in a depletion or weak-inversion situation, so that the photopotential Vph and/or the photocurrent Iph and/or the quadrature optoelectrochemical impedance component Zoq varies significantly with the bias potential Vb.
Thus, xe2x80x9cVbi corresponding approximately to the potential at the point of inflectionxe2x80x9d should be understood, according to the invention, to be within a range of uncertainty of xc2x10.5 volts.
The ordinate of the point of inflection of these Vph, Iph or Zoq =f(Vb) curves is advantageously chosen as a reference parameter to be kept constant within the context of this regulating by adjusting the Vb. The reference point is in practice given by the maximum value reached by Vph, Iph or Zoq in each of the characteristic sigmoidal curves.
In other words, Vph,max, Iph,max and Zoq,max correspond to the maximum value of the photopotential, the photocurrent and the quadrature optoelectrochemical impedance component, respectively, when the semiconductor is in a strong-inversion situation.
Insofar as the determination of the set value Vbi involves taking into account the abscissa and the ordinate of the point of inflection of at least one of the Vph or Zoq or Iph=f(Vb) curves, it is envisaged in this second mode of impementation to construct at least one of said curves, preferably as described above in the case of the first mode of impementation.
Version (i) of step d according to this second mode of impementation consists in monitoring xcex94Vfb by measuring the variation in Vph, Iph or Zoq after calibration. This amounts to constructing the Vph and/or Iph and/or Zoq=f(t) curve and to assuming that any xcex94Vph, xcex94Iph or xcex94Zoq variation corresponds to a xcex94Vfb variation. Version (i) can be used in the case of a moderate variation in Vfb, making it possible to establish a simple relationship (for example a linear relationship) between xcex94Vph and xcex94Vfb.
In practice, it is preferred to use version (ii) of step d according to the second mode of implementing the method according to the invention. In this version, Vph,xc2xdmax is kept constant by adjusting the bias potential Vb of the structure, preferably by means of electronic regulation.
The regulation xcex94Vb corresponds to the xcex94Vfb that it is desired to measure.
According to an advantageous arrangement of the invention, the electronic regulation that may be used within the context of version (ii) of taking xcex94Vb into account is carried out in the following manner:
Vph is detected;
optionally, the signal Vph is amplified;
optionally, this signal is rectified so as to give a DC signal Vxe2x80x2ph;
this DC signal Vxe2x80x2ph is compared with a reference signal U corresponding approximately to the value of Vxe2x80x2ph,xc2xdmax (the ordinate of the point of inflection of the curve Vxe2x80x2ph=f(Vb), i.e. Vxe2x80x2ph,xc2xdmax);
the difference between Vxe2x80x2ph and U, namely xcex94(Vxe2x80x2phxe2x88x92U), is obtained;
optionally, xcex94(Vxe2x80x2phxe2x88x92U) is amplified;
the optionally amplified xcex94(Vxe2x80x2phxe2x88x92U) is applied between Sc and CL as a complement of Vb;
xcex94(Vxe2x80x2phxe2x88x92U) is recorded.
The advantage of such a regulating procedure stems from the fact that the electrically imposed potential is directly and linearly the image of the Vfb variations caused by the interactions on the surface of the dielectric Is. It is therefore independent of the properties of the structure and allows good sensitivity and excellent measurement reproducibility to be obtained.
According to the nomenclature adopted in the present description, Vph denotes both the primary photopotential Vph measured between Sc and CL and the Vxe2x80x2ph corresponding to the rectified and optionally amplified Vph.
With regard to the illumination step c, which is common to both the abovementioned modes of implementation, it should be pointed out that said illumination may be carried out opposite any region of the external faces of the Sc/Is-Pr structure, including, where appropriate, on its thickness, or even over the entire external surface of this structure.
In other words, the periodic illumination of the Sc/Is structure is therefore carried out opposite the external face or faces of the Sc, or opposite the external face or faces of the insulator Is or opposite all the external faces of the structure.
In the case of step c, the wavelength xcex of the illumination is preferably chosen so that it is greater than or equal to             λ      0        =                  1240                  E          ⁢                      xe2x80x83                    ⁢                      (                          band              ⁢                              xe2x80x83                            ⁢              gap              ⁢                              xe2x80x83                            ⁢              energy                        )                              ⁢              xe2x80x83            ⁢      nm        ,
and preferably between 100 and 3000 nm, E being the band gap energy of the Sc expressed in electron volts.
This threshold xcex0 depends on the nature of the semiconductor.
According to a third way of implementing the method according to the invention, the nonspecific responses maybe radically removed by employing a differential measurement in which at least one other reference structure Sc/Is is used in the affinity sensor, in which Is is not functionalized by Pr, and by monitoring the difference between the Vfb variations measured by the 2 sensors, as well as the variation in this difference.
In this third mode of impementation, the measurement structure Sc/Is-Pr and the reference structure Sc/Is are brought into contact with the same medium CL. They are biased in the same way, preferably by means of the same counterelectrode, a reference electrode not being necessary. Finally, in accordance with step c, they are exposed to the same modulated-light illumination.
Moreover, the means for interpreting the detected signals used in step d are chosen so as to make it possible to monitor the difference between the flat band potentials Vfb0 and Vfb1 specific to the two structures, Sc/Is and Sc/Is-Pr, respectively.
This arrangement makes it possible to obtain a very precise and continuous measurement of the variation in Vfb1 of the functionalized structure Sc/Is-Pr, by overcoming the spurious effects which may occur and are associated with parameters other than the pairing, and in particular other than the PN/cPN hybridization.
A fourth way of implementing the method according to the invention is one in which, in a prior step a0, various regions on the surface of a layer of insulator Is of the same structure Sc/Is are functionalized by several probes Pr which differ in the nature of the cPNs of which they are composed and then, using this structure, the CL containing one or various PN substrates is analyzed:
by illuminating, in succession, the regions of the surface of the Is, said regions each being functionalized by the same Pr (identical cPNs), the nature of the Prs varying from one region to another;
and by measuring and interpreting the xcex94Vfb variations.
This fourth mode of impementation corresponds to the multiple detection of substrates, particularly PN substrates, of different kinds, within the genetics field. The multiple detection according to the invention makes it possible to use simple and rapid methods for detecting viral disorders and genetic disorders. It is also conceivable to make checks of histocompatibility, as well as the mapping of heterogeneous populations of polynucleotides, for example of genomes. This furthermore constitutes a tool for studying gene mutation, gene expression or genome sequencing.
The advantage of multiple detection according to this fourth mode of implementation lies in the methodological and structural simplicity of the corresponding method and the corresponding device.
The advantage of multiple detection according to this fourth mode of implementation lies in the methodological and structural simplicity of the corresponding method and the corresponding device.
In a manner common to the third and fourth modes of implementation mentioned above, namely differential measurement and, multiple detection respectively, it should be pointed out that the, step of measuring xcex94Vfb directly or indirectly may be carried out according to the procedure described in the case of the first mode of implementation in one or other of these weak- or strong-illumination versions, and likewise according to the procedure specific to the second mode of implementation, whether it is version (i) or version (ii) that is adopted.
The Invention is a measurement method founded on the recognition of the variation in the flat band potential (xcex94Vfb) of a structure Sc/Is-Pr having a sensitive region made of probes Pr based on recognition biological substances RBS (e.g. cPNs) capable of reacting specifically with electrically charged biological substances CBS (e.g. PNs) in a medium CL.
Advantageously, the substrates to be analyzed are PNs, preferably chosen from the following list: nucleotides, oligonucleotides, polynucleotides, nucleic acids (DNA or RNA) as well as similar materials and mixtures of said substrates.
According to the invention, the probes Pr employed in the method are means of specific recognition, particularly of unlabeled cPNs, that is to say those not carrying means of revealing the PN/cPN pairing (fluorescence, colorimetry or radioactivity).
Although preference is given to biological substances of the polynucleotide-sequence type (RNA, DNA, gene, plasmid or any other genetic material), this does not in any way exclude other biological substances of the antigen, hapten or antibody type or, more generally, any species of a couple formed by a biological macromolecule and its specific complement. As examples of specific-pairing couples or pairs, mention may be made of: antigen/antibody, hapten/antibody, cDNA/DNA, cRNA/RNA, poly (dT)/mRNA eukaryotic or lectin-glyco-conjugated, cell (or microorganism) marker/cell (or microorganism), HcG/tissue receptor and T3/TGB (thyroxin binding protein).
The method according to the invention firstly involves the immobilization of at least one type of these reactive biological species cPN in order to form the probe or probes Pr. The immobilization may be carried out directly on the insulating layer or using an intermediate material (for example spacing compounds) fastened to the insulator Is and capable of receiving, by physical bonding (e.g. adsorption/absorption) and/or chemical bonding (e.g. covalent bond), the specific biological ligands cPN.
According to the invention, it is completely conceivable to provide one of the heterospecific probes Pr formed from biological species CPN of different types, which are capable of reacting with their complementary species.
The conductive liquid medium CL may be any buffer solution compatible with the biological substances PN in question. This liquid medium CL advantageously has a conductivity equivalent to that of an aqueous NaCl solution having a concentration that can range from 0.005M to 3M and preferably about 0.1M.
The pH of the liquid medium may be between 0 and 12, preferably between 6 and 8, so as to favor the pairings by bioaffinity. The harshness of the liquid medium may also be adjusted in order to favor hybridization.
The nonspecific interactions, by exchange of ions or by hydrophobic interactions, may be eliminated completely or partly using a buffer of suitable ionic force (e.g. Tris-HCl/Tris-base).
Advantageously, the measurement temperature may be between 0 and 50xc2x0 C. It is controlled more precisely so as to favor the biochemical reactions in question.
The subject of the invention is also a device for implementing the method as described above, said device being one which comprises:
at least one affinity sensor formed by at least one structure Sc/Is-Pr in which the probe or probes Pr comprise ligands RBS suitable for being specifically hybridized with the biological substances CBS to be analyzed, these being contained in the conductive liquid medium CL, by causing a charge-effect phenomenon giving rise to the xcex94Vfb variations of the Sc;
means for biasing the Sc with respect to the CL;
means for illuminating the Sc of the sensor;
means for measuring the photopotential Vph or the photocurrent Iph;
means for converting the detected signals into Vfb variations;
and means for calculating and interpreting the xcex94Vfb variations in terms of the identification and/or quantitative determination of the biological substances CBS.
Roughly speaking, this device therefore comprises:
firstly, one or more simple semiconductor multilayer structures that can be likened to measurement electrodes;
secondly, a source of modulated light for illuminating this or these structures; and
thirdly, electronic peripheral means for electrical biasing, for measurement, for calculation and for interpretation.
The measurements of the photopotential Vph and/or the in-phase optoelectrochemical impedance component Zop and/or quadrature optoelectrochemical impedance component Zoq are carried out in an open-circuit configuration, that is to say the measurement circuit must have a very high load impedance.
The measurements of the photocurrent Iph are advantageously carried out in a closed-circuit configuration (short circuit).
These technical means involve conventional technologies fully controlled by those skilled in the art.
Furthermore, this device benefits from a low manufacturing cost. It may be easily produced on an industrial scale and offers high reliability and good measurement reproducibility.
In addition, the already miniaturized, or indeed xe2x80x9cminiaturizablexe2x80x9d, character of the affinity sensors constituting the device opens up promising future prospects, especially for certain in vivo or in vitro applications.
Preferably, but nonlimitingly, the measurement electrode (that is to say the multilayer structure Sc/Is-Pr) of the affinity sensor of the device is formed by at least one wafer of Sc, preferably made of silicon, covered on one of its faces with at least one layer of insulator, Is, preferably silica, fastened to the surface of which is at least one sensitive probe Pr comprising at least one biospecific pairing ligand, preferably polynucleotides cPN for specific recognition by hybridization, this sensor also having at least one ohmic contact allowing connection of the structure Sc/Is-Pr, especially to the biasing means and/or the means for measuring Vph.
The device furthermore comprises this structure Sc/Is-Pr, at least one auxiliary electrode, possibly provided on the layer Is of the structure Sc/Is-Pr.
Advantageously, the layer of blocking dielectric material Is is for example, a layer of oxide and/or of nitride or any other inorganic or organic material of small thickness, preventing or limiting the faradaic phenomena. This structure is associated with a conductive liquid medium CL containing the substances CBS to be detected or identified or quantitatively determined.
In practice, the silicon chosen as semiconductor is of the n or p type and is moderately doped, preferably to a level of 1015 to 1019 cmxe2x88x923, preferably 1016 cmxe2x88x923. The thickness of the silicon wafer is, for example, between 0.01 and 2 mm.
The insulating layer Is advantageously consists of silica or silicon nitride. Its function is to cancel out any faradaic process which might disturb the measurements, by means of spurious electrical signals. This layer Is also prevents the difficulties associated with possible corrosion of the semiconductor material by the conductive medium CL in contact with it. The thickness of this layer Is is between 1 and 500 nanometers, preferably between 1 and 50 and even more preferably about 10 nanometers.
The dielectric layer Is supports the probes cPN-Pr, which probes constitute what might be called a bioreceptive membrane in contact with the medium CL.